High fat:low carbohydrate relative to high carbohydrate:low fat feeding is associated with a marked reduction in insulin's ability to stimulate glucose transport in the isolated rat adipose cell. Since insulin appears to enhance glucose transport by triggering a rapid and reversible translocation of glucose transport systems from a large intracellular pool to the plasma membrane, the effects of high fat:low carbohydrate feeding on this pool and the translocation process in the isolated rat adipose cell have been examined. With high fat:low carbohydrate feeding, insulin's ability to stimulate 3-0-methylglucose transport in intact cells and to increase the concentration of glucose transport systems in the plasma membrane fraction prepared from these cells is decreased by 55% and 41%, respectively. At the same time, the basal concentration of glucose transport systems in the low-density microsomal membrane fraction prepared from cells of high fat:low carbohydrate-fed rats is decreased by 47%. Thus, the reduced glucose transport response of the rat adipose cell to insulin with high fat:low carbohydrate feeding appears to be the consequence of a relative depletion of glucose transport systems in the intracellular pool.